ABCMdb

PMID: 17495464

Kraus C, Reis A, Naehrlich L, Dotsch J, Korbmacher C, Rauh R
Functional characterization of a novel CFTR mutation P67S identified in a patient with atypical cystic fibrosis.
Cell Physiol Biochem. 2007;19(5-6):239-48., [PubMed]

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0 ABCC7 p.Pro67Ser Original Paper Cell Physiol Biochem 2007;19:239-248 Accepted: December 04, 2006Cellular PhysiologyCellular PhysiologyCellular PhysiologyCellular PhysiologyCellular Physiology and Biochemistrand Biochemistrand Biochemistrand Biochemistrand Biochemistryyyyy Copyright (c) 2007 S. Karger AG, Basel Fax +41 61 306 12 34 E-Mail karger@karger.ch www.karger.com (c) 2007 S. Karger AG, Basel 1015-8987/07/0196-0239$23.50/0 Accessible online at: www.karger.com/cbp Functional Characterization of a Novel CFTR Mutation P67S Identified in a Patient with Atypical Cystic Fibrosis Cornelia Kraus1 , André Reis1 , Lutz Naehrlich2 , Jörg Dötsch2 , Christoph Korbmacher3 and Robert Rauh3 1 Institute of Human Genetics, University of Erlangen-Nuremberg, 2 University Hospital for Children and Adolescents, University of Erlangen-Nuremberg, 3 Institute of Cellular and Molecular Physiology, University of Erlangen-Nuremberg Prof. Login to comment 2 ABCC7 p.Pro67Ser 6, D-91054 Erlangen (Germany) Tel. +49 9131 85 22301, Fax +49 9131 85 22770 E-Mail christoph.korbmacher@physiologie2.med.uni-erlangen.de Key Words CFTR • P67S mutation • Cystic fibrosis • Pancreatic insufficiency • Xenopus laevis oocytes • Chloride channel • Electrophysiology • Surface expression Abstract Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Login to comment 6 ABCC7 p.Pro67Ser Full length sequencing of the patient`s CFTR gene revealed a homozygous C to T transition at nucleotide position 331 (CCT>TCT), which results in a P67S amino acid substitution. Login to comment 10 ABCC7 p.Pro67Ser Expression of P67S-CFTR resulted in functional CFTR chloride channels. Login to comment 14 ABCC7 p.Pro67Ser Our findings indicate that the P67S mutation reduces CFTR chloride channel function by reducing channel surface expression. Login to comment 36 ABCC7 p.Pro67Ser ABCC7 p.Pro67Ser However, full sequencing of the CFTR gene revealed a homozygous mutation, that leads to a substitution of a serine for a proline at position 67 (P67S) in the amino acid chain of the CFTR protein. Login to comment 37 ABCC7 p.Pro67Ser The aim of this study was to investigate the functional effect of P67S in the Xenopus laevis oocyte expression system using whole-cell current measurements and a chemiluminescence assay to assess channel surface expression. Login to comment 38 ABCC7 p.Pro67Ser We found that P67S largely reduces cAMP stimulated CFTR chloride currents and channel surface expression without altering the ion selectivity of the channel. Login to comment 39 ABCC7 p.Pro67Ser Our findings indicate that P67S represents a class II mutation of CFTR associated with abnormal or reduced channel trafficking. Login to comment 85 ABCC7 p.Pro67Ser In a second step a P67S mutation was inserted into the wild-type CFTR clone (forward: AGC TGG CTT CAA AGA AAA ATT CTA AAC TCA TTA ATG CCC TTC G; reverse: CGA AGG GCA TTA ATG AGT TTA GAA TTT TTC TTT GAA GCC AGC T). Login to comment 86 ABCC7 p.Pro67Leu In the same way a P67L mutation was generated (forward: GCT GGC TTC AAA GAA AAA TCT TAA ACT CAT TAA TGC CCT TCG; reverse: CGA AGG GCA TTA ATG AGT TTA AGA TTT TTC TTT GAA GCC AGC). Login to comment 101 ABCC7 p.Pro67Leu ABCC7 p.Pro67Ser Defolliculated stage V-VI oocytes were injected (Nanoject automatic injector, Drummond, Broomall, PA) with 0.25 ng cRNA of wild-type CFTR or P67S mutant or P67L mutant. Login to comment 123 ABCC7 p.Pro67Ser ABCC7 p.Pro67Ser Sequencing of exon 3 revealed a homozygous C to T transition at nucleotide position 331 (CCT>TCT), which results in a substitution of a serine for a proline at position 67 (P67S). Login to comment 127 ABCC7 p.Pro67Ser Therefore, we conclude that P67S is a novel CFTR mutation. Login to comment 129 ABCC7 p.Pro67Ser To test the functional relevance of the mutation, we heterologously expressed wild-type and P67S mutant CFTR in Xenopus laevis oocytes. Login to comment 132 ABCC7 p.Pro67Ser ABCC7 p.Pro67Ser Sequencing analysis of CFTR exon 3 at the level of genomic DNA revealed a homozygous C>T transition at nucleotide position c.331 in the patient. This transition resulted in the substitution of a serine for a proline at position 67 (p.P67S). Login to comment 134 ABCC7 p.Pro67Ser Substitution of serine for proline at position 67 reduces CFTR whole-cell currents As previously described [21] we used a combination of 1mM IBMX and 1µM forskolin (IBMX/Fsk) to elevate intracellular cAMP and activate whole-cell CFTR Cl- currents (ΔIIBMX/Fsk ) in the oocyte expression system. Login to comment 135 ABCC7 p.Pro67Ser Figure 2A shows typical whole-cell current recordings obtained from an oocyte expressing wild-type CFTR (upper trace) and from an oocyte expressing the P67S mutant CFTR (lower trace) at a holding potential of -60 mV. Login to comment 138 ABCC7 p.Pro67Ser These findings indicate that expression of P67S mutant CFTR results in functional CFTR chloride channels. Login to comment 139 ABCC7 p.Pro67Ser However, the CFTR whole-cell currents activated by IBMX/Fsk were much smaller in oocytes expressing the P67S mutant CFTR than in matched oocytes expressing wild-type CFTR. Login to comment 141 ABCC7 p.Pro67Ser The P67S mutant CFTR channel is functional but at a largely reduced level. Login to comment 142 ABCC7 p.Pro67Ser A: Representative whole-cell current traces recorded at a holding potential of -60 mV from an oocyte expressing wild-type CFTR (upper trace) or the P67S mutant CFTR (bottom trace). Login to comment 145 ABCC7 p.Pro67Ser Voltage step protocols for current-voltage (I-V) plots were performed at times indicated by asterisks, but the complete current responses were omitted from the continuous current trace for clarity B: Average I-V plots of IBMX/Fsk induced CFTR whole-cell currents (ΔIIBMX/Fsk ) were obtained from experiments as shown in A using matched oocytes expressing wild-type CFTR (circles) or P67S mutant CFTR (diamonds) (N=7, n=52 per group). Login to comment 147 ABCC7 p.Pro67Ser The steeper slope of the wild-type CFTR I-V curve compared to the slope of the P67S mutant CFTR I-V curve indicates a reduced CFTR whole-cell conductance in oocytes expressing the mutant channel. Login to comment 148 ABCC7 p.Pro67Ser C: Representative Western blot analysis to detect CFTR protein expression in oocytes injected with 2ng of cRNA coding for wild-type (wt) or P67S mutant CFTR or in non-injected control oocytes (n. Login to comment 153 ABCC7 p.Pro67Ser ΔGIBMX/ Fsk averaged 31.8 ± 4.3 µS (n = 52) in P67S mutant expressing oocytes and was significantly lower than ΔGIBMX/ Fsk in wild-type CFTR expressing oocytes (131.9 ± 12.5 µS, n = 52, p<0.0001). Login to comment 154 ABCC7 p.Pro67Ser These results demonstrate that the P67S mutation does not abolish but largely reduces CFTR chloride channel function in the oocyte system. Login to comment 156 ABCC7 p.Pro67Ser Thus, the reduced channel function of the P67S mutant compared to wild-type CFTR is unlikely to be caused by reduced protein synthesis of the mutant channel. Login to comment 159 ABCC7 p.Pro67Ser Since the P67S mutation is not localized in the vicinity of channel regions thought to contribute to the pore of the CFTR channel or its gating mechanism [3], we hypothesized that the mutation was likely to affect channel surface expression. Login to comment 160 ABCC7 p.Pro67Ser To investigate a possible effect of the P67S mutation on channel surface expression, we used HA-tagged CFTR constructs and a chemiluminescence assay as described previously to measure CFTR surface expression and whole-cell currents in parallel [21]. Login to comment 161 ABCC7 p.Pro67Ser In three independent experiments using different batches of oocytes we found a large reduction of the surface expression of the P67S mutant channel compared to wild-type CFTR. Login to comment 162 ABCC7 p.Pro67Ser Our results indicate that a reduced surface expression of the P67S mutant CFTR can fully explain the reduction of ΔGIBMX/Fsk observed in oocytes expressing the mutant channel (figure 3). Login to comment 163 ABCC7 p.Pro67Ser The P67S mutation does not alter the anion selectivity of CFTR It is well known that mutations in CFTR can influence the anion selectivity of the channel [26]. Login to comment 164 ABCC7 p.Pro67Ser Therefore, we wanted to test whether the P67S mutation affects the anion selectivity of the IBMX/Fsk induced whole-cell conductance. Login to comment 170 ABCC7 p.Pro67Ser Surface expression of the P67S mutant CFTR channel is reduced. Login to comment 171 ABCC7 p.Pro67Ser Surface expression (open columns) and IBMX/Fsk stimulated whole-cell conductance (closed columns) of HA-tagged wild-type and P67S mutant CFTR channels were assessed in matched groups of oocytes. Login to comment 181 ABCC7 p.Pro67Ser These results indicate that the P67S mutation does not alter the anion selectivity of CFTR. Login to comment 182 ABCC7 p.Pro67Leu ABCC7 p.Pro67Ser ABCC7 p.Pro67Ser The previously reported P67L mutation has a similar effect on CFTR function as the P67S mutation As shown above the P67S mutation reduces ΔGIBMX/ Fsk by decreasing the surface expression of the mutated CFTR channels. Login to comment 185 ABCC7 p.Pro67Ser A: Typical whole-cell recordings of two-electrode voltage-clamp measurements of a wild-type CFTR (top) and a P67S mutant (bottom) expressing oocyte after activation with IBMX/Fsk. Login to comment 189 ABCC7 p.Pro67Ser Please note the different current scales for wild-type CFTR and the P67S mutant. Login to comment 193 ABCC7 p.Pro67Ser There was no difference in the whole-cell conductance sequence of wild-type CFTR (open bars, n=3) and P67S mutant CFTR (grey bars, n=3). Login to comment 194 ABCC7 p.Pro67Leu same position in the CFTR protein has previously been described, P67L, and was reported to cause a dominant mild phenotype of CF [29], i.e. if a patient is heterozygous for this mutation, he suffers from a mild CF even when the mutation on the other allele is known to cause severe CF (e.g. ΔF508). Login to comment 196 ABCC7 p.Pro67Leu ABCC7 p.Pro67Ser Therefore, we decided to investigate the P67L mutation to compare its effect with the effect of the P67S mutation. Login to comment 197 ABCC7 p.Pro67Leu ABCC7 p.Pro67Ser In two different batches of oocytes we found that ΔGIBMX/Fsk was similarly decreased in P67S CFTR (30.2 ± 2.8 µS, n=9) and P67L CFTR (14.3 ± 4.1 µS, n=9) expressing oocytes compared to ΔGIBMX/Fsk of wild-type CFTR (157.0 ± 24.4 µS, n=9, see figure 5) expressing oocytes. Login to comment 200 ABCC7 p.Pro67Leu ABCC7 p.Pro67Ser IBMX/forskolin activated whole-cell conductance (ΔGIBMX/Fsk ) of wild-type, P67S, and P67L CFTR. Login to comment 201 ABCC7 p.Pro67Leu ABCC7 p.Pro67Ser Experiments were essentially performed as described in figure 2 to determine ΔGIBMX/Fsk in matched oocytes from two different batches expressing either wild-type CFTR, P67S or P67L CFTR (N=2, n=9,** p<0.01, *** p<0.001). Login to comment 203 ABCC7 p.Pro67Ser The following findings of this study support the hypothesis that this mutation is indeed pathophysiologically relevant: 1) Expression of the P67S mutant CFTR in Xenopus laevis oocytes resulted in a CFTR whole-cell conductance that averaged only 24 % of that of wild-type CFTR. Login to comment 204 ABCC7 p.Pro67Ser 2) The surface expression of the P67S mutant was also largely reduced compared to that of wild-type CFTR. Login to comment 205 ABCC7 p.Pro67Leu ABCC7 p.Pro67Ser 3) The mutation P67L, a previously described dominant mild form of CFTR [29], reduced the CFTR whole-cell conductance to about the same level as the P67S mutation. Login to comment 206 ABCC7 p.Pro67Ser Therefore we believe that P67S is a novel CFTR mutation which in homozygous patients can cause a mild phenotype of CF characterized by abdominal pain and pancreatic insufficiency in our patient. Login to comment 207 ABCC7 p.Pro67Ser The combination of a reduced CFTR whole-cell conductance, decreased channel surface expression, and normal protein expression level indicates that the P67S mutation causes a defect in channel trafficking and corresponds to a class II mutation [16]. Login to comment 211 ABCC7 p.Pro67Ser Interestingly, the P67S mutation appeared to reduce channel surface expression even more than CFTR whole-cell conductance (fig. 3). Login to comment 213 ABCC7 p.Pro67Ser However, from structural considerations it is unlikely that the P67S mutation affects the channel pore or the gating mechanism [3, 30]. Login to comment 218 ABCC7 p.Pro67Ser Thus, the P67S mutation is located distant from the channel pore or from domains likely to be involved in channel gating. Login to comment 219 ABCC7 p.Pro67Ser Moreover, our finding that the P67S mutation does not affect the anion selectivity of the channel supports our conclusion that the mutation does not affect the channel pore. Login to comment 221 ABCC7 p.Pro67Ser In conclusion it is unlikely that the P67S mutation has a major effect on channel properties like single channel conductance or open probability. Login to comment 223 ABCC7 p.Pro67Leu ABCC7 p.Pro67Leu This P67L mutation was found to confer a dominant mild effect, i.e. compound heterozygotes carrying the P67L mutation on one allele and a severe CFTR mutation (e.g. ΔF508) on the other allele have a mild phenotype. Login to comment 224 ABCC7 p.Pro67Leu Gilfillan et al. [29] reported that none of the P67L compound heterozygotes they investigated showed consistently raised sweat chloride concentrations and 77% of the 13 patients investigated were pancreatic sufficient. Login to comment 225 ABCC7 p.Pro67Leu Another study [31] reported one patient with a negative sweat test and pancreatic sufficiency, who was also heterozygous for P67L. Login to comment 226 ABCC7 p.Pro67Leu To our knowledge no functional data have yet been reported for the P67L mutant. Login to comment 227 ABCC7 p.Pro67Leu ABCC7 p.Pro67Ser In our study we found that the P67L mutant had a similar inhibitory effect on the CFTR whole-cell conductance as the P67S mutant. Login to comment 228 ABCC7 p.Pro67Ser These findings confirm the functional relevance of the P67 residue and support our conclusion that the newly identified P67S mutation can lead to a mild CF phenotype. Login to comment 231 ABCC7 p.Pro67Leu ABCC7 p.Pro67Ser Thus, in the oocyte expression system the inhibitory effect of the ΔF508 mutation seems to be rather similar to that observed in the present study for the P67S and P67L mutant channels. Login to comment 236 ABCC7 p.Pro67Leu ABCC7 p.Pro67Ser Thus, the temperature sensitivity of the ΔF508-CFTR may explain its more severe phenotype compared to the P67S or the P67L mutations. Login to comment 237 ABCC7 p.Pro67Leu ABCC7 p.Pro67Ser In any case, the mild CF phenotype of patients with the P67S or the P67L mutations suggests that the residual function of these mutant channels is sufficient to prevent severe symptoms. Login to comment 239 ABCC7 p.Pro67Ser The P67S mutation in our patient was found on the background of a M470V polymorphism of CFTR. Login to comment 241 ABCC7 p.Pro67Ser ABCC7 p.Pro67Ser It is conceivable that the combination of V470 and P67S leads to a mild phenotype of CF whereas the combination of M470 and P67S shows a normal phenotype. Login to comment 242 ABCC7 p.Pro67Ser However, to clarify this question it would be necessary to identify persons with a M470 P67S genotype and to test for symptoms of CF. Login to comment 243 ABCC7 p.Pro67Ser In addition we have to consider that the phenotype of the P67S-CFTR mutation may be influenced by modifier genes or other factors [43]. Login to comment 245 ABCC7 p.Pro67Ser The P67S mutant channels can be stimulated by exposure to IBMX/Fsk and have normal anion selectivity. Login to comment 247 ABCC7 p.Pro67Ser The residual function of the P67S mutant channel is consistent with the mild CF phenotype of the affected patient. Login to comment